Lipids make up a group of biological substances which have in common their insolubility in water and high solubility in organic solvents (e.g., chloroform). They have several important biological roles, such as serving as components of membranes, providing fuel and functioning as highly concentrated energy stores.
Phospholipids make up one of the four major groups of membrane lipids; glycolipids, cholesterol and glyceride derivatives (e.g., triglycerides, diglycerides, monoglycerides and component fatty acids) are the others. Phospholipids are derived from the three-carbon alcohol, glycerol, or from a more complex alcohol, sphingosine. Those derived from glycerol are called phosphoglycerides and occur almost exclusively in cell membranes. A phosphoglyceride is comprised of a glycerol backbone, two fatty acid chains and a phosphorylated alcohol. One of the primary hydroxyl groups of glycerol is esterified to phosphoric acid; a polar head group, an alcohol represented by X--OH, is esterified to the phosphoric acid through its hydroxyl group. Commonly occurring alcohol components of phosphoglycerides are choline, ethanolamine, serine, glycerol and inositol. The resulting phospholipids are called, respectively, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), diphosphatidylglycerol (PG) and phosphatidylinositol (PI). The two remaining hydroxyl groups of the glycerol backbone are esterified to fatty acids, which may be saturated or unsaturated. These hydrocarbon tails are nonpolar in nature.
Phospholipids containing sphingosine (or a related base) as their backbone are called sphingolipids and are components of plant and animal cell membranes. These phospholipids have three characteristic components: a fatty acid; sphingosine or a related derivative; and a polar head group. In higher animals, sphingomyelins are the most commonly occurring sphingolipids. They contain phosphorylcholine or phosphorylethanolamine as the polar head groups and exhibit physical properties similar to those of PC and PE.
Because they have a polar head group and nonpolar hydrocarbon tails, phospholipids are amphiphilic (also called amphipathic) or polar lipids. In water or other aqueous medium, the polar head groups exhibit their affinity for water and the hydrocarbon chains avoid water. This can be achieved in at least two ways: by forming micelles or by forming a lipid bilayer (or biomolecular sheet). Short-chain phospholipids (those with fatty acids of fewer than 9 carbon atoms) form micelles when dispersed in aqueous solutions. R. J. M. Tausk et al., Biophysics and Chemistry, 1:175-183(1974); R. J. M. Tausk et al., Biophysics and Chemistry, 2:53-63 (1974). In a micelle, the polar head groups are on the surface and the hydrocarbon chains aligned inside the structure. On average, the terminal methyl group of the fatty acyl chains is at the center of the hydrophobic sphere, ellipsoid or cylindrical structure.
The lipid bilayer configuration is formed rapidly when long-chain phospholipids are placed in water. Because of their amphiphilic nature, the lipid molecules arrange themselves spontaneously into the bilayer configuration. In the bilayer, the polar head groups are at the two surfaces of the bilayer and the hydrophobic hydrocarbon chains are sequestered in the bilayer interior.
Lipid bilayers are held together by several noncovalent interactions and exposure of hydrocarbon chains to the aqueous medium is minimized. Lipid bilayers tend to close on themselves so that there will be no ends, and thus no hydrocarbon chains exposed to the aqueous medium. This results in the formation of a lipid-enclosed aqueous compartment. In addition, the bilayers tend to be self-sealing; a hole in the bilayer is energetically unfavorable.
The term liposome or lipid vesicle refers to the lipid bilayers and the encapsulated aqueous compartment. When long-chain phospholipids (e.g., PC, PS, PE, phosphatidic acid) are dispersed in an aqueous solution, they spontaneously form large multilamellar structures. These structures consist of many concentric layers of lipid, which have aqueous phases interspersed between the bilayers. B. E. Ryman and D. A. Tyrrell, "Liposomes--Bags of Potential," In: Essays in Biochemistry, P. N. Campbell and R. D. Marshall (ed.), Academic Press, London, 49-98. (1980). These multilamellar vesicles (MLVs) may also be formed by the addition of an aqueous phase to a dry lipid film (e.g., egg phosphatidylcholine), followed by gentle agitation.
A wide variety of lipids has been used in liposome preparations (Table I). Usually PC or sphingomyelin is the major lipid and small amounts of other species (e.g., charged amphiphiles such as stearylamine, dicetylphosphate) are added.
TABLE I ______________________________________ Lipids Used in Liposome Preparation Lipid Comments ______________________________________ Egg Phosphatidylcholine Most commonly used major component Dipalmitoyl-PC Synthetic fully satur- ated phosphatidyl- choline Distearoyl-PC Less permeable to aqueous phase than egg PC Sphingomyelin Often preferred to egg PC in immunological studies; improves liposome stability in vivo Cholesterol Reduces permeability of egg PC vesicles; maximum incorporation of 50 mol % with phospholipids Stearylamine Imparts net positive charge; not naturally occurring; may be toxic to cells Phosphatidic acid Imparts net negative charge Phosphatidylserine Imparts net negative charge Cardiolipin Antigenic lipid used in immunological applications Phosphatidylethanolamine Does not form enclosed vesicle on its own; useful for coupling materials to the external surface of liposomes; substituted derivatives are used in immunological studies Lysophosphatidylcholine Increases liposome permeability; may enhance liposome fusion with cells ______________________________________
Unilamellar vesicles--which are aqueous compartments surrounded by only one shell of lipid bilayer--may be formed from the multibilayers (MLVs). Several methods have been developed for forming unilamellar vesicles from MLVs. F. Szoka and D. Papahadjopoulos, Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes), Annual Review of Biophysics and Bioengineering, 9:467-508. (1980). Each, however, either requires special equipment or involves nonphospholipid material (e.g., organic solvents, detergents) and is a multistep process. One of these methods involves sonication--agitation by high-frequency sound waves--of an aqueous suspension of a suitable lipid. D. Papahadjopoulos and H. K. Kimelberg, Progress in Surface Science, W. G. Davison (ed.), Pergamon Press, 4:139-221, (1973). Alternative methods for producing unilamellar vesicles include reverse phase evaporation from organic solvent, F. Szoka, Jr. and D. Papahadjopoulos, Proceedings of the National Academy of Sciences, U.S.A. 75:4194-4198, (1978); detergent dialysis or dilution, J. Brunner et al., Biochimica et Biophysica Acta, 455:322-331, (1976); and pressure/mechanical filtration. R. Hamilton et al., Journal of Lipid Research, 21:981-992, (1980); M. C. Farmer and B. P. Gaber, Biophysics, 45:41a, (1984). These methods were developed for producing unilamellar vesicles from MLVs containing PCs and have not been extensively used with other phospholipids. Often, the vesicles produced by altering the MLVs using these methods are not stable; tend to aggregate or fuse; and are likely to release their contents.
Gains and Hauser report a method of forming small unilamellar vesicles from a solution of dilauroyl phosphatidic acid or mixtures of dilauroyl phosphatidic acid and PC in chloroform/methanol. N. Gains and H. Hauser, Characterization of Small Unilamellar Vesicles Produced in Unsonicated Phosphaticid Acid and Phosphatidylcholine-Phosphatidic Acid Dispersions by pH Adjustment, Biochimica et Biophysica Acta, 731:31-39 (1983). The method involves formation of a film from such a solution, dispersing the film in water or an aqueous solution containing sodium chloride and sulfate ions; raising the pH of the solution by titration with sodium hydroxide; and subsequent lowering of the pH. It is claimed that as the pH of the solutions is raised, vesicles are formed because the phosphate group of the phosphatidic acid molecules becomes ionized. This method is not generally applicable as a means of producing unilamellar vesicles, however. It requires the presence of the anionic phospholipid phosphatidic acid and cannot be used to produce PC or PE unilamellar vesicles. The pH shifts necessary to form the vesicles according to this method limit its use because many proteins and other substances to be encapsulated in the vesicles will be unstable to or adversely affected by such shifts.
Substances may be entrapped or incorporated into liposomes in a number of ways and therefore liposomes have great potential in a wide variety of fields. For example, in medicine there has long been a need for biodegradable vehicles or carriers for therapeutic materials because such materials may be toxic unless they are incorporated into a carrier or delivery vehicle; are degraded by the body before they reach their target; or are diffused throughout the body, instead of being delivered to a desired target site. MLVs may be useful for this delivery or protection purpose but are usually too large for effective dispersal. Alternatively, MLVs may be processed by one or more of the existing methods to produce unilamellar vesicles, which are more suitable for these purposes.
Current methods of producing unilamellar vesicles, however, are far less than satisfactory because they require specialized equipment, involve nonphospholipid materials which might contaminate or alter the resulting lipid vesicle - entrapped substance combination and/or are time consuming because they require multiple steps. The unilamellar vesicles produced by these methods are also often or typically less than satisfactory because of their instability (which often depends on vesicle curvature and size), tendency to aggregate and release of the entrapped material. Of the methods available for making small unilamellar vesicles, only two--sonication and extrusion through a French press--give good encapsulation efficiencies. However, both methods require specialized equipment and several steps. In methods which involve detergent removal, much of the drug to be encapsulated is likely to be lost before the vesicles are formed.